Method and compositions for the treatment and detection of endothelin-1 related kidney diseases

ABSTRACT

The present application relates to methods of treating HIV-associated nephropathy (HIVAN) and/or focal segmental glomerulosclerosis (FSGS) using endothelin-1 (ET-1) antagonists. The application further relates to a composition for the treatment of HIVAN and/or FSGS. A kit for detecting the presence of ET-1 or ET-1-associated biomarker in a biological sample is also disclosed.

This application is a Continuation of U.S. application Ser. No.17/094,175, filed Nov. 10, 2020, which is a continuation applicationSer. No. 13/906,074, filed May 30, 2013, now U.S. Pat. No. 10,866,249,which is a Continuation of U.S. application Ser. No. 13/162,010, filedJun. 16, 2011, now U.S. Pat. No. 9,255,931, which claims priority fromProvisional Application No. 61/344,289, filed on Jun. 24, 2010. Theentirety of the aforementioned applications is incorporated herein byreference.

This application was made with U.S. Government support under Grant No.5P20RR011104, awarded by the NIH/NCDD/Clinical Research Centers. Thus,the U.S. Government may have certain rights in this application.

FIELD

This application relates generally to medical treatment and detectionand, in particular, to the treatment and detection of endothelin-1(ET-1) related nephropathy, such as focal segmental glomerulosclerosis(FSGS) and HIV-associated nephropathy (HIVAN).

BACKGROUND

HIV associated nephropathy (HIVAN) affects predominantly AfricanAmericans with HIV-1 infection, and is the most common cause of renalfailure in HIV positive patients. The pathological features of HIVANinclude collapsing focal segmental glomerulosclerosis (FSGS) withmicrocystic tubular dilation and interstitial inflammation. Highlyactive antiretroviral therapy (HAART) appears to limit the progressionto end-stage renal disease (ESRD), but is not successful in all cases(Schwartz et al., J. Am. Soc. Nephrol. 16:2412-2420, 2005), and longterm use of antiretrovirals can be toxic to the kidneys (Izzedine etal., Am. J. Kidney Dis. 45:804-817, 2005). Untreated HIV positiveindividuals who develop HIVAN can progress rapidly to ESRD within 6-12months (Szczech et al., Kidney Int 66:1145-1152, 2004).

There is no standard treatment of HIVAN and randomized controlledclinical trials have not been performed (Yahaya et al., CochraneDatabase of Systematic Reviews, 2009). Most treatment regimens have beenbased on therapies used to treat the symptoms of kidney disease. Thesedrugs have included angiotensin converting enzyme inhibitors, steroidsand cyclosporine; however, conclusive data concerning their efficacy isnot available (Yahaya et al. supra). There exists a need to discovermore effective pharmacological agents to treat HIVAN and reduce theprogression to ESRD.

There exists a need for more effective pharmacological agents fortreating HIVAN and other kidney diseases, and for identifying patientsbenefiting from such treatment. Accordingly, the present applicationprovides such compositions and methods for use.

SUMMARY

One aspect of the present invention relates to a method of treatingHIV-associated nephropathy and/or focal segmental glomerulosclerosis ina subject by administering to said subject an effective amount of anET-1 antagonist, wherein said subject has increased amount ofcirculating plasma ET-1 compared to other HIV positive patients andhealthy controls.

Another aspect of the present invention relates to a method formonitoring the effectiveness of a treatment for HIV-associatednephropathy (HIVAN) and/or focal segmental glomerulosclerosis (FSGS) ina subject. In one embodiment, the method comprises the steps of: (a)performing a first measurement of ET-1 and/or an ET-1-associatedbiomarker in a first biological sample from the subject, (b) performinga second measurement of ET-1 and/or an ET-1-associated biomarker insecond biological sample from the subject, and (c) determining anefficacy of the treatment based on the results of (a) and (b). In arelated embodiment, step (a) is performed prior to the initiation of thetreatment and step (b) is performed after the initiation of thetreatment.

Another aspect of the present invention relates to a method fordiagnosis of HIVAN and/or FSGS in a subject. The method comprises thesteps of comparing a level of an ET-1-associated biomarker in abiological sample obtained from the subject to a threshold level; andmaking a diagnosis based on a result from the comparing step. In arelated embodiment, the method further comprises the step of determiningthe level of the ET-1-associated biomarker in the biological sampleobtained from the subject.

Another aspect of the present invention relates to a composition for thetreatment of HIV-associated nephropathy and/or focal segmentalglomerulosclerosis. In one embodiment, the composition comprises an ET-1antagonist and a pharmaceutically acceptable carrier.

Another aspect of the present invention relates to a kit for detectingthe presence of ET-1 or ET-1 associated biomarker in a biologicalsample.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram showing that ET-1 plasma concentrations in healthycontrols and HIV infected patients.

FIG. 2 is a composite of picture panels showing immunohistochemistry ofET-1 in kidney biopsies. Immunohistochemical localization of ET-1 inkidney tissue from donor nephrectomies (panels a and b) compared withrenal biopsies from patients with primary (idiopathic) focal segmentalglomerulosclerosis (FSGS) (panels c and d) or HIV infection (panels eand f). Original magnification of Panels a, c and e=100×. Originalmagnification of Panels b, d and f=200×. G=glomerulus.

FIG. 3 is a diagram showing preproET-1 (PpET-1) mRNA expression inhealthy Caucasian and African American controls (healthy, n=9), HIVpositive Caucasians (White HIV+, n=4), HIV positive African Americans(AA HIV+, n=7), and HIVAN patients (n=6).

FIG. 4 is a diagram showing ECE-1 mRNA expression in healthy Caucasianand African American controls (healthy, n=9), HIV positive Caucasians(White HIV+, n=4), HIV positive African Americans (AA HIV+, n=7), andHIVAN patients (n=6).

FIG. 5 is a diagram showing a SNP analysis correlating differences inplasma ET-1 expression and T allele frequencies between the followingpatient groups: HIVAN, AA HIV positive, non-AA HIV positive, healthy AA,and healthy non-AA.

DETAILED DESCRIPTION

The following detailed description is presented to enable any personskilled in the art to make and use the invention. For purposes ofexplanation, specific nomenclature is set forth to provide a thoroughunderstanding of the present invention. However, it will be apparent toone skilled in the art that these specific details are not required topractice the invention. Descriptions of specific applications areprovided only as representative examples. Various modifications to thepreferred embodiments will be readily apparent to one skilled in theart, and the general principles defined herein may be applied to otherembodiments and applications without departing from the scope of theinvention.

In case of conflict, the present specification, including definitions,will control. Following long-standing patent law convention, the terms“a” “an” and “the” mean “one or more” when used in this application,including in the claims.

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about”. Accordingly, unless indicated to the contrary, thenumerical parameters set forth in this specification and attached claimsare approximations that can vary depending upon the desired propertiessought to be obtained by the presently disclosed subject matter. As usedherein, the term “about,” when referring to a value or to an amount ofmass, weight, time, volume, concentration or percentage is meant toencompass variations of in some embodiments ±20%, in some embodiments±10%, in some embodiments ±5%, in some embodiments ±1%, in someembodiments ±0.5%, and in some embodiments ±0.1% from the specifiedamount, as such variations are appropriate to perform the disclosedmethod.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which the presently disclosed subject matter belongs.Although any methods, devices, and materials similar or equivalent tothose described herein can be used in the practice or testing of thepresently disclosed subject matter, representative methods, devices, andmaterials are now described.

The term “direct ET-1-associated biomarker” as used herein, refers toproteins and/or polynucleotides directly linked to ET-1 expression orET-1 activity. Exemplary ET-1-associated biomarkers include, but are notlimited to proteins, nucleic acids, antibodies, single nucleotidepolymorphisms (SNPs) and/or probes therefrom capable of specificallydetecting the presence or expression (e.g., in a biological sample froma subject) of preproendothelin (ppET-1), proendothelin-1 (pET-1), bigET-1, ET-1, and derivatives therefrom.

The term “indirect ET-1-associated biomarker” as used herein, refers toproteins and/or polynucleotides indirectly correlated with ET-1expression or ET-1 activity. Exemplary ET-1-associated biomarkersinclude, but are not limited to endothelin converting enzyme (ECE),cystatin C; creatinine, including albumin-creatinine ratio (ACR); andother measures characteristic of kidney diseases

The term “ET-1-associated biomarker” as used herein, refers to bothdirect ET-1-associated biomarker and indirect ET-1-associated biomarker.

The term “ET-1 level” refers to an expression level corresponding to anyone of the direct ET-1-associated biomarkers, included, but not limitedto preproendothelin (ppET-1), proendothelin-1 (pET-1), big ET-1, ET-1,and derivatives therefrom.

Nephropathy including but not limit to, glomerulopathy, glomerulitis,glomerulonephritis, glomerulonephrosis, tubulitis. HIVAN, FSGS, druginduced nephropathy, nephron loss, hyperfiltration, chronicpyelonephritis, Type I RPG/Type II hypersensitivity, Goodpasture'ssyndrome, Type II RPG/Type III hypersensitivity, Lupus (DPN),IgA/Berger's nephropathy, Wegener's granulomatosis, Microscopicpolyangiitis, RTA (RTA 2), Fanconi syndrome, Bartter syndrome, Gitelmansyndrome, Liddle's syndrome RTA (RTA 1), diabetes insipidus(Nephrogenic), renal papilla, renal papillary necrosis, majorcalyx/pelvis, hydronephrosis, pyonephrosis, reflux nephropathy, acutetubular necrosis, interstitial nephritis, pyelonephritis, danubianendemic, familial nephropathy, renal failure, acute renal failure,chronic renal failure, uremic pericarditis, uremia, renal arterystenosis, renal Ischemia, hypertensive nephropathy, renovascularhypertension, analgesic nephropathy, renal osteodystrophy, nephroptosis,and Abderhalden-Kaufmann-Lignac syndrome.

The term “control subject” refers to a comparative subject providing ameans for determining whether treatment with an ET-1 antagonist iswarranted, for determining the effectiveness of a given treatment, orfor determining whether a given subject is at risk for developing adisease characterized by increased ET-1 levels as further discussedherein. A “control subject” can refer to a healthy or diseased subjectreceiving a mock treatment (e.g., saline alone), a healthy subjectreceiving the same treatment as a diseased patient or subject underassessment for disease risk, a diseased subject known to exhibitincreased ET-1 levels, or combination thereof. Additionally, a healthyor diseased subject may be of the same or different ethnicity as thesubject under assessment for disease risk and/or treatment.

Endothelin-1 (ET-1) is the most potent vasoconstrictor known. It issynthesized by many cell types, including vascular endothelial cells,glomerular mesangial cells, renal epithelial cells, neutrophils,monocytes and macrophages. ET-1 is generated from precursor peptides viaa two-step proteolytic pathway. Transcription of a gene on chromosome 6generates an mRNA encoding the 212-amino acid peptidepre-proendoethelin-1 (preproET-1 or ppET-1), which once translated, isstripped of its signal sequence and secreted into the cytoplasm asproET-1 (Kirby et al., Br. J. Pharmacol., 153:1105-1119, 2008). ProET-1is further cleaved by a furin-like endopeptidase to a biologicallyinactive 38-amino acid precursor, “big ET-1”, which circulates in plasmaat low concentration. Removal of a further 17 COOH-terminal residues,classically but not exclusively by a membrane-bound metalloproteinase,ET-converting enzyme (ECE), results in formation of the mature,biologically active 21-amino acid pET-1 peptide, the most potentvasoconstrictor known.

ET-1 binds to ET-1_(A) and ET-1_(B) receptors. In the kidney, ET-1causes diminished blood flow and glomerular filtration rate. ET-1 iselevated in many renal diseases. ET-1 induces vasoconstriction, cellproliferation and extracellular matrix formation, all features ofchronic renal disease. Mesangial cells possess receptors for ET-1 andcan also produce ET-1, which in turn stimulates mesangial cellproliferation and extracellular matrix hypersecretion (Sorokin, et al.,Am. J. Physiol Renal Physiol 285:F579-F589, 2003). Studies have shownincreased production of urinary and plasma ET-1 concentrations inpatients with idiopathic FSGS and IgA nephropathy (Chen et al., J Clin.Lab Anal. 15:59-63-16, 2001). Both resting and stimulated macrophagesproduce and release ET-1 (Ebihara et al., Periton. Dialy. Intern.17:595-601, 1997). Macrophages are a major constituent of theinterstitial infiltrate that is frequently found in close proximity tosclerotic glomeruli and plays a role in the development of HIVAN (Bodiet al., Am. J. Kidney Dis. 24:762-767, 1994).

Normotensive and hypertensive African American adults and adolescentsproduce more ET-1 than their Caucasian counterparts at rest and inresponse to stress (Treiber et al., Hypertens. 35:722-725, 2000; Treiberet al., Psychosomatic Medicine 64:707-713, 2002). HIVAN affectspredominantly African Americans with HIV-1 infection, and is the mostcommon cause of renal failure in AIDS patients (Winston et al., Mt.Sinai J. Med. 65:27-32, 1998). Sickle cell anemia, a disease mostprevalent in individuals of African descent, also is associated withincreased ET-1 levels. Plasma ET-1 is elevated in sickle cell anemiapatients as well as those undergoing acute vaso-occlusive sickle crisiswhen compared to African Americans without sickle cell disease (Rybickiet al., Blood 92:2594-2596, 1998; Graido-Gonzalez et al., Blood92:2551-2555, 1998). In addition, African Americans have a higherincidence of chronic kidney disease (Boone, Nephrol. Nurs. J.27:597-600, 2000) that may be related to the MYH9 gene (Kao et al., Nat.Genet. 40:1185-1192, 2008); Singh et al., Am. J Kidney Dis. 54:732-740,2009).

Treatment Methods

One aspect of the present invention relates to a method of treatingHIV-associated nephropathy (HIVAN) and/or focal segmentalglomerulosclerosis (FSGS) comprising administering into a subject aneffective amount of an ET-1 antagonist. In a preferred embodiment, thesubject exhibits increased levels of ET-1 compared to other HIV positivepatients or healthy controls. The method is particularly suited forethnic groups and/or patients exhibiting higher than normal ET-1 levels,including, but not limited to HIVAN patients, FSGS patients, AfricanAmerican HIV-positive patients, HIV-positive patients, and patients withother kidney diseases, including, but not limited to IgA nephropathy,diabetic nephropathy, Sickle cell nephropathy, and renovascularhypertension.

In one embodiment, the ET-1 antagonist binds to one or more of ET-1,ET-2, ET-3, or a peptide derivative thereof. In another embodiment, theET-1 antagonist binds to an ET receptor, such as ET-1_(A). In eithercase, the ET-1 antagonist preferably prevents or reduces the interactionbetween ET-1 to an ET receptor. As such, the ET-1 antagonist may be asmall molecule, such as an organic compound, or a large molecule, suchas a protein, peptide, antibody, soluble ET receptor, nucleic acid,oligonucletide, small interferring RNA (siRNA), and the like.

Exemplary ET-1 antagonists may selected from the group consisting ofsitaxentan, ambrisentan, atrasentan, BQ-123, bosentan and tezosentan.ET-1 antagonists are well know to those skilled in the art, and mayinclude any of those disclosed in U.S. Patent Application PublicationNo. 2010/0022568 to Clozel et al., the disclosure of which isincorporated by reference herein.

An ET-1 antagonist may be administered by any route suitable forreducing ET-1 levels in the subject compared to a control subject,including, but not limited to intravenous, intramuscular, nasal,transdermal, transmucosal, buccal, sublingual, oral, subdermaladministration, or combinations thereof.

In certain embodiments, the ET-1 antagonist is infused locally into thekidney. The ET-1 antagonist may be introduced into the kidney byintravenous, intrarterial, or retrograde infusion. In one embodiment,the vis infused through the vene renalis. In another embodiment, theET-1 antagonist is infused through the superior mesenteric artery. Inyet another embodiment, the ET-1 antagonist is infused through aretrograde catheter into the pyelic cavity. Since only a relativelysmall amount of ET-1 antagonist is needed for the kidney infusion, theantagonist-related toxicity is reduced. In yet another embodiment, thekidney is perfused with the ET-1 antagonist, i.e., the ET-1 antagonistenters the kidney through the vene renalis or the superior mesentericartery, and is collected through the superior mesenteric artery or venerenalis. Since the leftover ET-1 antagonist does not enter the bloodcirculation, a large amount of ET-1 antagonist may be used for theperfusion. In addition, a close-circuit perfusion allows constantexposure to virus over an extended period of time (e.g., 10-60 minutes).

The ET-1 antagonist may be administered to a subject exhibiting an ET-1level or ET-1-associated biomarker level of at least about 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 100% higher than the corresponding level in a controlsubject. Alternatively, the ET-1 antagonist may be administered to asubject exhibiting an ET-1 level or ET-1-associated biomarker level ofat least about at least about 1.5 fold, 2-fold, 2.5-fold, 3-fold,4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, or100-fold higher than the corresponding level in a control subject.Formulations and routes of administering the ET-1 antagonists in theclaimed treatment methods are detailed below.

Determining Effectiveness of a Treatment

In another aspect, the present invention provides a method formonitoring the effectiveness of a treatment for HIVAN, FSGS, or otherkidney-related disease in a subject. In one embodiment, the methodcomprises the steps of (a) performing a first measurement of ET-1 and/oran ET-1-associated biomarker in a first biological sample from thesubject, (b) performing a second measurement of ET-1 and/or anET-1-associated biomarker in second biological sample from the subject,and (c) determining the effectiveness of the treatment based on theresults of steps (a) and (b). In a preferred embodiment, step (a) isperformed prior to the initiation of the treatment and step (b) isperformed after the initiation of the treatment.

The ET-1-associated biomarker may be a direct ET-1-associated biomarkeror an indirect ET-1 associated biomarker. In one embodiment, theET-1-associated biomarker is a direct ET-1-associated biomarker selectedfrom the group consisting of preproendothelin (ppET-1), proendothelin-1(pET-1), big ET-1, ET-1, and derivatives therefrom. In anotherembodiment, the ET-1-associated biomarker is an indirect ET-1-associatedbiomarker selected from the group consisting of endothelin convertingenzyme (ECE), cystatin C, creatinine, albumin-creatinine ratio (ACR);and other measures characteristic of kidney diseases. The biologicalsample may be derived from blood, plasma, urine, saliva or tissue.

An exemplary method for detecting the presence of the ET-1-associatedbiomarker in a biological sample involves obtaining a biological samplefrom a test subject and contacting the biological sample with a bindingagent capable of binding to and facilitating detection of theET-1-associated biomarker. The binding reagent may be an antibody,protein, peptide, polynucleotide, oligonucleotide, fragment thereof, orconjugate thereof.

A preferred agent for detecting an ET-1-associated biomarker peptide orprotein, such as ET-1, is an antibody or a portion thereof that bindsspecifically the biomarker. Preferred agents for detectingET-1-associated mRNAs include oligonucleotide probes capable ofhybridizing to an ET-1-associated mRNA in a biological sample or offacilitating reverse transcriptase-based assays for detection of ET-1associated biomarkers in the biological sample, such as ET-1 mRNA.

Detection of the ET-1-associated biomarker may be facilitated throughthe use of secondary binding agents and detection schemes well known tothose skilled in the art. Assays including but not limited toimmunoassay and RT-PCR analyses may be used to quantify the amount ofexpression or activity of the ET-1-associated biomarker in thebiological sample. Such quantification may be useful, for example, todetermine the progression or severity of HIVAN, FSGS, or a given kidneydisease, or to monitor the effectiveness of a treatment therefore usingthe ET-1 antagonists of the present invention. Immunoassay and RT-PCRmethodologies for use in the present invention include those describedin the Examples below and in U.S. Patent Application Publication No.2010/0029560 to Donald et al., the disclosure of which is incorporatedby reference herein.

Diagnosis Method

In another aspect, the present invention provides a method for diagnosisof HIVAN, FSGS, or other kidney diseases in a subject. In oneembodiment, the method comprises the steps of comparing a level of anET-1-associated biomarker in a biological sample obtained from thesubject to a threshold level, and making a diagnosis based a result fromthe comparing step. In one embodiment, the biological sample is a plasmasample and the ET-1-associated biomarker is ET-1. In another embodiment,the threshold level is a plasma ET-1 level at about 4 pg/ml and a testsubject is deemed to have a high risk of HIVAN and/or FSGS, if theplasma ET-1 level in the subject is higher than the threshold level. Inanother embodiment, the threshold level is a plasma ET-1 level at about4.5 pg/ml and a test subject is deemed to have a high risk of HIVANand/or FSGS, if the plasma ET-1 level in the subject is higher than thethreshold level. In another embodiment, the level of the ET-1-associatedbiomarker is the ppET-1 mRNA level in peripheral blood derivedmacrophages that are harvested from the subject and cultured in thepresence of IV Nef. In another embodiment, the level of theET-1-associated biomarker is the ppET-1 mRNA level in peripheral bloodderived macrophages that are harvested from the subject and cultured inthe presence of 10 ng/ml HIV Nef for four hours and the threshold valueis about 5 copies of ppET-1 mRNA per 10⁶ copies of 18s rRNA, asdetermined by reverse transcriptase-real time quantitative PCR. Inanother embodiment, the method further comprises the step of determininga level of an ET-1-associated biomarker in a biological sample for thesubject.

In another embodiment, the ET-1-associated biomarker is selected fromthe group consisting of preproendothelin (ppET-1), proendothelin-1(pET-1), BIG ET-1, ET-1, derivatives therefrom, endothelin convertingenzyme (ECE), cystatin C, creatinine, albumin-creatinine ratio (ACR) orother measures characteristic of kidney diseases. The biological samplecan be blood, plasma, urine, saliva or tissue.

Pharmaceutical Compositions

Another aspect of the present invention relates to a composition for thetreatment of HIVAN and/or FSGS. In one embodiment, the compositioncomprises an ET-1 antagonist and a pharmaceutically acceptable carrier.In one embodiment, the ET-1 antagonist binds to one or more of ET-1,ET-2, ET-3, or a peptide derivative thereof. In another embodiment, theET-1 antagonist binds to an ET receptor, such as ET-1_(A). In eithercase, the ET-1 antagonist preferably prevents or reduces the interactionbetween ET-1 to an ET receptor. As such, the ET-1 antagonist may be asmall molecule, such as an organic compound, or a large molecule, suchas a protein, peptide, antibody, soluble ET receptor, nucleic acid,oligonucletide, small interferring RNA (siRNA), and the like.

Exemplary ET-1 antagonists may selected from the group consisting ofsitaxentan, ambrisentan, atrasentan, BQ-123, bosentan and tezosentan.ET-1 antagonists are well know to those skilled in the art, and mayinclude any of those disclosed in U.S. Patent Application PublicationNo. 2010/0022568 to Clozel et al., the disclosure of which isincorporated by reference herein.

A pharmaceutical composition in accordance with the present inventionincludes a pharmaceutically acceptable carrier. As used herein, a“pharmaceutically acceptable carrier” is intended to include any and allsolvents, solubilizers, fillers, stabilizers, binders, absorbents,bases, buffering agents, lubricants, controlled release vehicles,diluents, emulsifying agents, humectants, lubricants, dispersion media,coatings, antibacterial or antifungal agents, isotonic and absorptiondelaying agents, and the like, compatible with pharmaceuticaladministration. The use of such media and agents for pharmaceuticallyactive substances is well-known in the art. See e.g., A. H. KibbeHandbook of Pharmaceutical Excipients, 3rd ed. Pharmaceutical Press,London, UK (2000). Except insofar as any conventional media or agent isincompatible with the active agent(s), use thereof in the compositionsis contemplated. Supplementary agents can also be incorporated into thecompositions.

In addition, the pharmaceutical composition may additionally includeother bioactive agents, such as angiotensin-converting enzyme (ACE)inhibitors, used in conjunction with the ET-1 antagonists of the presentinvention. Examples of ACE inhibitors include, but are not limited to,sulfhydryl-containing agents such as captopril and zofenopril;dicarboxylate-containing agents, such as enalapril, ramipril, quinapril,perindopril, lisinopril, benazepril, imidapril, zofenopril; andphosphonate-containing agents, such as fosinopril.

A pharmaceutical composition of the invention may be formulated forcompatibility with any route of administration suitable for reducingET-1 levels or ET-1 activity in a patient. Exemplary routes ofadministration include intravenous, intramuscular, nasal, transdermal,transmucosal, buccal, sublingual, oral, subdermal, or combinationthereof. Solutions or suspensions used for such formulations may includeany of the following components, including a sterile diluent such aswater for injection, saline solution, fixed oils, polyethylene glycols,glycerine; propylene glycol or other synthetic solvents; antibacterialagents such as benzyl alcohol or methyl parabens; antioxidants such asascorbic acid or sodium bisulfate; chelating agents such asethylenediaminetetraacetic acid; buffers such as acetates, citrates orphosphates and agents for the adjustment of tonicity such as sodiumchloride or dextrose. pH can be adjusted with acids or bases, such ashydrochloric acid or sodium hydroxide. The pharmaceutical compositioncan be enclosed in ampoules, disposable syringes or multiple dose vialsmade of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the injectable composition should be sterile and should be fluidto the extent that easy syringability exists. It must be stable underthe conditions of manufacture and storage and must be preserved againstthe contaminating action of microorganisms such as bacteria and fungi.The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyetheylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequited particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activeET-1 antagonist and/or other additional bioactive agents in apharmaceutically effective amount in an appropriate solvent with one ora combination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active agent(s) into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying, which can yield a powder of the active,ingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activeagent(s) can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the activeagent(s) in the fluid carrier are applied orally and swished andexpectorated or swallowed. Pharmaceutically compatible binding agents,and/or adjuvant materials can be included as part of the composition.The tablets, pills, capsules, troches and the like can contain any ofthe following ingredients, or compounds of a similar nature: a bindersuch as microcrystalline cellulose, gum tragacanth or gelatin; anexcipient such as starch or lactose, a disintegrating agent such asalginic acid, Primogel, or corn starch; a lubricant such as magnesiumstearate or Stertes; a glidant such as colloidal silicon dioxide; asweetening agent such as sucrose or saccharin; or a flavoring agent suchas peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the active agent(s) may be deliveredin the form of an aerosol spray from pressured container or dispenserwhich contains a suitable propellant, e.g., a gas such as carbondioxide, or a nebulizer.

Systemic administration can also be by transmucosal, buccal, ortransdermal means. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art, andinclude, for example, for transmucosal administration, detergents, bilesalts, and fusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the bioactive agent(s) may be formulatedinto ointments, salves, gels, or creams as generally known in the art.

The ET-1 antagonists can also be prepared in the form of suppositories(e.g., with conventional suppository bases such as cocoa butter andother glycerides) or retention enemas for rectal delivery.

The ET-1 antagonist formulations may be prepared using carriersprotecting the ET-1 antagonist against rapid elimination from the body,such as a controlled release formulations, including implants,nanoparticles, microparticles, and microencapsulated delivery systems.Controlled release nanoparticles are described in U.S. PatentApplication Publication No. 2007/0224280 to Lillard et al., thedisclosure of which is incorporated by reference herein. Biodegradable,biocompatible polymers can be used, such as ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, andpolylactic acid. Methods for preparation of such formulations will beapparent to those skilled in the art. The materials can also be obtainedcommercially from e.g. Alza Corporation and Nova Pharmaceuticals, Inc.Liposomal formulations (including liposomes targeted to infected cellswith monoclonal antibodies to viral antigens) can also be used aspharmaceutically acceptable carriers. Liposomal formulations can beprepared according to methods well known to those skilled in the art,for example, as described in U.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein includesphysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activeagent(s)calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active agent(s) and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of formulating such active agent(s) for thetreatment of individuals.

Toxicity and therapeutic efficacy of the active agent(s) of the presentinvention can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., for determining the LD50(the dose lethal to 50% of the population) and the ED50 (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio LD50/ED50. Active agents exhibiting largetherapeutic indices are preferred. While active agents exhibiting toxicside effects may be used, care should be taken to design a deliverysystem that targets such agents to the site of affected tissue in orderto minimize potential damage to uninfected cells and, thereby, reduceside effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofthe active agents lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any active agentsused in the method of the invention, the therapeutically effective dosecan be estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test agent which achieves a half-maximal inhibition of symptoms) asdetermined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

The pharmaceutical compositions can be included in a kit, container,pack, or dispenser, together with instructions for administration.

Kits

The invention also encompasses kits for detecting the presence of anET-1-associated biomarkers in a biological sample, the kit comprisingreagents for measuring an ET-1-associated biomarker in a biologicalsample. Preferably, the reagents include at least one binding agent inthe form of a binding agent capable of binding to and facilitatingdetection of the ET-1-associated biomarker. The binding agent may be anantibody, protein, peptide, polynucleotide, oligonucleotide, fragmentthereof, or conjugate thereof.

A preferred binding agent for detecting an ET-1-associated biomarkerpeptide or protein, such as ET-1, is an antibody or a portion thereofthat binds specifically the biomarker. Preferred binding agents fordetecting ET-1-associated mRNAs include oligonucleotide probes capableof hybridizing to an ET-1-associated mRNA in a biological sample or offacilitating reverse transcriptase-based assays for detection of ET-1associated biomarkers in the biological sample, such as ET-1 mRNA (asfurther described below).

Binding agents for detection of ET-1 associated biomarkers may beprepared by methods known in the art. The kit may contain means fordetermining the amount of the ET-1 associated biomarker protein or mRNAin the sample; and means for comparing the amount of ET-1-associatedbiomarker in the sample with a control or standard. The binding agentcan be packaged in a suitable container.

The kit may further include a label or packaging insert including adescription of the components or instructions for use. Exemplaryinstructions may include, for example, instructions for collectingbiological sample(s), for detecting ET-1-associated biomarker(s), andfor interpreting the results. The kits described above may additionallyinclude liquids suitable for resuspending any of the kit reagents, alongwith one or more container(s) for collecting biological sample(s),and/or centrifuge filter(s) for isolating components in the biologicalsample(s).

The present invention is further illustrated by the following exampleswhich should not be construed as limiting. The contents of allreferences, patents and published patent applications cited throughoutthis application, as well as the Figures are incorporated herein byreference.

Example 1: Detection of ET-1 and ET-1-Associated Markers Study Subject

Patients with biopsy-proven HIVAN were also recruited from variousacademic medical centers in the United States. Information on thesepatients was limited to diagnosis and race. Patient charts were reviewedfor HIV and HIVAN status. HIV sero positive patients who presented withproteinuria, >2 g/day, and elevated blood creatinine levels, >2.0 mg/dL,were either biopsied for HIVAN confirmation, or were diagnosedpresumptively as having HIVAN after eliminating factors such ashypertension, hepatitis and diabetes. HIV seropositive patients lackingrenal disease were enrolled in the study and designated “no history ofkidney disease” if: (1) they had no proteinuria (dipstick negative ortrace proteinuria <0.15 g/d), (2) their serum creatinine was <2 mg/dLduring their previous two clinic visits within a year and at the time oftheir blood donation; (3) their CD4⁺ T cells were greater than 200/mm³;and (4) they had no AIDS defining illness. Healthy controls wereseronegative for HIV infection, had no history of kidney disease andwere not hypertensive.

A total of 239 patients were enrolled in this study: HIVAN (n=64),African American HIV seropositive without kidney disease (n=75),Caucasian HIV seropositive without kidney disease (n=48), and healthycontrols (n=134; 75 African American and 59 Caucasian). Human subjectsamples were collected in accordance with protocols approved by theInstitutional Review Board and the Human Subjects Research Committee atall institutions, and informed consent was obtained from all patientsand healthy volunteers.

Blood Collection

One 10 ml EDTA tube was used to collect whole blood from patients. Thetubes were centrifuged, and plasma was collected and frozen at −80° C.until analysis for ET-1.

ET-1 Quantification

ET-1 levels present in plasma were determined by colorimetric ELISA.ET-1 was extracted from the plasma samples using acetone: 1N HCl:water(40:1:5) Cross reactivity for big ET-1 was less than 1%, while ET-2 andET-3 had a 45% and 14% cross reactivity respectively as determined bythe manufacturer. The plates were measured using a microplate reader setat 450 rim. Samples were assayed in triplicate.

Immunohistochemistry

Twelve biopsy samples from 4 HIVAN, 4 FSGS and 4 healthy controls weredeparafinized and stained for ET-1. Mouse monoclonal ET-1 was used asthe primary antibody and a goat anti mouse IgG conjugated to horseradishperoxidase as the secondary antibody. Samples were blinded and scoredfor staining intensity, 0-4 scale, in podocytes, renal tubules, thevasculature and interstitial cells.

Peripheral Blood Macrophage Culture

Peripheral blood mononuclear cells (PBMCs) from HIVAN (n=6), AfricanAmerican HIV⁺ (n=7), Caucasian HIV⁺ (n=4), healthy African American(n=4), and healthy Caucasian (n=5) controls were isolated by Histopaquedensity gradient separation. Macrophages were purified from the PBMCpopulation by adherence to plastic. Briefly, PBMCs were washed in HanksBalanced Salt Solution without Ca⁺⁺ and Mg⁺⁺ (HBSS), then resuspended inIscoves Modified Dulbecco Medium (IMDM) with 10% male human serum. ThePBMCs were plated in 6 well microtiter plates at a cell density of 1×10⁵cells per well and incubated at 37° C. for 24 hrs. The plates werewashed twice with HBSS and the adherent cells were removed with coldtrypsin-EDTA. The recovered monocytes were washed and plated in 6 wellcluster plates at a density of 1×105 cells/ml in IMDM plus 10% humanserum, and incubated for 5-7 days for macrophage maturation. The finalcell preparations contained approximately 80% monocytes/macrophages asdetermined by nonspecific esterase staining. Cell viability was greaterthan 98%, as assessed by trypan blue exclusion.

Cells were stimulated with either 100 ng/ml LPS (E. coli serotype0111::B4) as a positive control, 10 ng/ml baculovirus-derived HIV-1gpl20_(BaL) (NIH AIDS Research and Reference Reagent Program), 10 ng/mlnon-myristylated E. coli-derived recombinant HIV Nef (rHIV Nef), ormedia only. The rHIV Nef was tested for LPS content by the limulusamebocyte assay and found to be free of contamination. Total RNA wasisolated from both resting and stimulated macrophages at 4 hours, usingTri-Reagent and frozen at −80° C.

Reverse Transcriptase-Real Time Quantitative PCR (RT qPCR)

Total RNA was reverse transcribed into cDNA using the Ominiscript®Reverse Transcriptase. The mixture was incubated for 60 mins at 37° C.One pg of RNA was converted to cDNA, of which 500 ng was used for realtime qPCR. In brief, 2 pl of each primer was added to 25 μl of a 2×QuantiTect™ SYBR Green PCR master mix in RNase-free water. cDNA wasadded to the mixture. Real time cycler conditions were: 15 min at 95° C.(PCR initial activation step), 40 cycles of 15 s at 94° C.(denaturation), 30 s at 55° C. (annealing), and 30 s at 72° C.(extension). Specific primer sequences are listed in Table 1. Values forthe amplified gene products were obtained through normalizing the geneof interest to the 18s rRNA concentration in each sample.

Statistics

For mRNA expression and protein analysis, statistical comparisons weremade using the non-parametric Mann-Whitney rank sum test forsignificance. For the SNP analyses, a Pearson's Chi-Square test was usedto determine significant differences in the frequencies of the T allelesbetween groups. Results are presented as the mean±SEM. Statisticalsignificance was defined as P<0.05 or less.

Example 2: Plasma ET-1 Levels in Different Patient Groups

As shown in FIG. 1, ET-1 in HIVAN patients was significantly elevated(4.66±0.20 pg/ml) compared to African American HIV positive (2.66±0.13pg/ml), Caucasian HIV positive patients (1.76±0.09 pg/ml) and healthyAfrican American (0.3±0.08 pg/ml) and Caucasian (0.44±0.12) controls(P<0.001 for all groups). All HIV positive patients had higherconcentrations of ET-1 when compared to controls without HIV infectionin the following order: HIVAN (n=65) >African American HIV⁺ patients(n=63) (P=<0.001) >Caucasian HIV⁺ patients (n=59) (P<0.001) >healthyAfrican American (n=77) and Caucasian (n=58) controls (P<0.001).Brackets denote significant differences between groups.

Example 3: ET-1 Expression in Kidney Biopsy Samples

As shown in FIG. 2, immunoperoxidase staining for ET-1 is negative inkidney tissue from a donor nephrectomy (panels a and b). A kidney biopsyfrom a patient with FSGS (panels c and d) exhibits increased content ofET-1 in parietal epithelium lining Bowman's space (large arrow in paneld), as well as visceral epithelial cells overlying a segmental scar(smaller arrow in panel d). Surrounding tubules also show increasedcytoplasmic expression of ET-1. A kidney biopsy from a patient infectedwith HIV (panels e and f) shows increased expression of ET-1 in tubules,similar to that in the FSGS sample. In contrast to FSGS, however,glomerular epithelial cells evidence only trace amounts of the protein.

Example 4: ppET-1 mRNA Levels in Different Patient Groups

Preproendothelin-1 (ppET-1) is the precursor polypeptide processed tobig ET-1 and then cleaved by ECE-1 to produce the active ET-1 peptide.Peripheral blood derived macrophage were cultured with media only(control), 100 ng/ml LPS, 10 ng/ml HIV Nef or 10 ng/ml HIV gp120 for 4hours. PpET-1 mRNA was detected using real time quantitative PCR andnormalized against 18s rRNA. All other significant differences aredenoted by the brackets. As shown in FIG. 3, all HIV positive patientshad significantly higher expression of ppET-1 mRNA when compared to thehealthy controls under all treatment conditions (P<0.003). ppET-1 mRNAexpression was highest in macrophages from HIVAN patients treated withHIV Nef when compared to all other groups (P<0.003). African AmericanHIV positive patients, including HIVAN patients, had significantlyhigher amounts of ppET-1 mRNA expressed in cells cultured in media ortreated with HIV Nef compared to HIV⁺ Caucasian patients (P<0.003). LPStreatment of HIVAN patients' macrophages stimulated significantly moreppET-1 (P<0.03) than LPS treatment of macrophages from Caucasian HIVpositive patients. In all cases, except cells from the healthy controls,LPS increased ppET-1 mRNA expression when compared to cells cultured inmedia only (P<0.02).

Example 5: ECE-1 mRNA Levels in Different Patient Groups

ECE-1 converts biologically inactive big ET-1 into the biologicallyactive ET-1 peptide. Thus, over-expression of ECE-1 can lead toincreased production of ET-1. Peripheral blood derived macrophages fromthe above described ethnic groups were treated as described above. ECE-1mRNA was detected using real time quantitative PCR and normalizedagainst 18s rRNA. Differences in macrophage ECE-1 mRNA expressionbetween ethnic and treatment groups are shown in FIG. 4. Brackets denotesignificant differences between groups.

HIV Nef induced the greatest amount of ECE-1 mRNA in African AmericanHIV positive macrophages, which was significantly higher than all theother groups (P<0.02). Macrophages from African American HIV positiveand HIVAN patients had significantly increased ECE-1 mRNA expressionwhen cultured in media only or treated with HIV Nef (P<0.02 and P<0.001,respectively) compared to the healthy controls and Caucasian HIVpositive patients. LPS treatment did not significantly increase ECE-1mRNA in any groups when compared to the other treatments. No significantdifferences were found between the Caucasian HIV positive patientsresponses and the healthy group. HIV gp120 did not induce any detectableECE-1 mRNA from any of the macrophages (data not shown).

Example 6: SNP Analysis in Different Patient Groups

Genomic DNA was isolated with DNAzol BD (Molecular Research Center) fromPBMCs. The ET-1 SNP was amplified using Hotstart Taq PCR (Qiagen) usingthe specific primers listed in Table 1. Pyrosequencing (Uppsala, Sweden)was performed using the ET-1 amplified PCR products. The forward primerwas biotinylated at the 5′ end for use in pyrosequencing. The nucleotidesequence was determined from the signal peaks in the pyrogram. Genotypeswere verified in a second laboratory using an ABI 3100 capillary nucleicacid sequencer (Applied Biosystems, Foster City, Calif.) and Taqmanprobes. DNA samples with discordant results were excluded from thestudy. The T allele frequencies between the following patient groups:HIVAN, AA HIV positive, non-AA HIV positive, healthy AA, and healthynon-AA are shown in FIG. 5.

TABLE 1 PCR Primer Sequences Gene Forward Primer Reverse Primer ppET- 1TGGACATCATTTGGGTCAACA TCTCTTGGACCTAGGCTTC (SEQ ID NO: 1) (SEQ ID NO: 2)ECE-1* AACCTTCGCCGAGGACAG GGCTGATTTTCGGGTTTC (SEQ ID NO: 3)(SEQ ID NO: 4) 18s rRNA* GTAACCCGTTGAACCCCATT CCATCCAATCGGTAGTAGCG(SEQ ID NO: 5) (SEQ ID NO: 6) Lys198Asn GTCGGAGACCATGAGAAACAGTTTTGAACGAGGACGCTGG GCG TCC (SEQ ID NO: 7) (SEQ ID NO: 8) *Designed byBeacon Designer (Premier Biosoft International, Palo Alto, CA)

The above description is for the purpose of teaching the person ofordinary skill in the art how to practice the present invention, and itis not intended to detail all those obvious modifications and variationsof it which will become apparent to the skilled worker upon reading thedescription. It is intended, however, that all such obviousmodifications and variations be included within the scope of the presentinvention, which is defined by the following embodiments. Theembodiments are intended to cover the components and steps in anysequence which is effective to meet the objectives there intended,unless the context specifically indicates the contrary.

What is claimed is:
 1. A method of treating HIV-associated nephropathy(HIVAN) or focal segmental glomerulosclerosis (FSGS), comprisingadministering to a mammalian subject an effective amount of acomposition comprising an ET-1 antagonist, wherein the subject has anincreased level of ET-1 compared to a control subject.
 2. The method ofclaim 1, wherein the control subject is an HIV positive subject.
 3. Themethod of claim 1, wherein the ET-1 antagonist is a small molecule thatbinds to ET-1 or ET-1_(A).
 4. The method of claim 1, wherein the ET-1antagonist is selected from the group consisting of sitaxentan,ambrisentan, atrasentan, BQ-123, bosentan and tezosentan.
 5. The methodof claim 1, wherein the ET-1 antagonist is administered by a localinfusion into a kidney of the subject.
 6. A method for diagnosis ofNephropathy in a subject, comprising: comparing a level of anET-1-associated biomarker in a biological sample obtained from thesubject to a threshold level; and making a diagnosis based a result fromthe comparing step.
 7. The method of claim 6, wherein the biologicalsample is blood, plasma, urine, saliva or tissue.
 8. The method of claim6, wherein the threshold level is a plasma ET-1 level at about 4 pg/mland the subject is deemed to have a high risk of HIVAN and/or FSGS, ifthe plasma ET-1 level in the subject is higher than the threshold level.9. The method of claim 6, wherein the threshold level is a plasma ET-1level at about 4.5 pg/ml and the subject is deemed to have a high riskof HIVAN and/or FSGS, if the plasma ET-1 level in the subject is higherthan the threshold level.
 10. The method of claim 6, wherein saidET-1-associated biomarker is ET-1.
 11. The method of claim 6, whereinthe level of the ET-1-associated biomarker is the ppET-1 mRNA level inperipheral blood derived macrophages that are harvested from the subjectand cultured in the presence of HIV Nef.
 12. The method of claim 6,further comprising determining the level of the ET-1-associatedbiomarker in the biological sample for the subject.
 13. The method ofclaim 6, wherein said nephropathy is select from the group consisting ofglomerulopathy, glomerulitis, glomerulonephritis, glomerulonephrosis,tubulitis, HIVAN, FSGS, Diabetic nephropathy, sickle cell nephropathy,renovascular hypertension, drug induced nephropathy, nephron loss,hyperfiltration, chronic pyelonephritis, Type I RPG/Type IIhypersensitivity, Goodpasture's syndrome, Type II RPG/Type IIIhypersensitivity, Lupus (DPN), IgA/Berger's nephropathy, Wegener'sgranulomatosis, Microscopic polyangiitis, RTA (RTA 2), Fanconi syndrome,Bartter syndrome, Gitelman syndrome, Liddle's syndrome, RTA (RTA 1),diabetes insipidus (Nephrogenic), renal papilla, renal papillarynecrosis, major calyx/pelvis, hydronephrosis, pyonephrosis, refluxnephropathy, acute tubular necrosis, interstitial nephritis,pyelonephritis, danubian endemic, familial nephropathy, renal failure,acute renal failure, chronic renal failure, uremic pericarditis, uremia,renal artery stenosis, renal ischemia, hypertensive nephropathy,renovascular hypertension, analgesic nephropathy, renal osteodystrophy,nephroptosis, and Abderhalden-Kaufmann-Lignac syndrome.
 14. The methodof claim 6, wherein said nephropathy is HIVAN or FSGS.
 15. A compositionfor the treatment of HIVAN or FSGS, said composition comprising an ET-1antagonist and a pharmaceutically acceptable carrier.
 16. Thecomposition of claim 15, wherein the ET-1 antagonist is a small moleculethat binds to ET-1 or ET-1_(A).
 17. The composition of claim 16, whereinthe ET-1 antagonist is selected from the group consisting of sitaxentan,ambrisentan, atrasentan, BQ-123, bosentan and tezosentan.
 18. Thecomposition of claim 16, wherein the composition is formulated for aroute of administration selected from the group consisting ofintravenous, intramuscular, nasal, transdermal, transmucosal, buccal,sublingual, oral, subdermal, or combination thereof.
 19. A kit fordetecting the presence of an ET-1-associated biomarker in a biologicalsample, wherein said kit comprises one or more reagents suitable formeasuring the ET-1-associated biomarker in the biological sample; andinstructions for using said kit to detect said ET-1-associatedbiomarker, wherein the biological sample is blood, plasma, urine, salivaor tissue.
 20. The kit of claim 19, wherein the reagents comprise anantibody, nucleic acid, or fragment thereof, wherein the antibody,nucleic acid, or fragment thereof specifically binds to theET-1-associated biomarker.